Three-dimensional (3D) printing has become one of the major innovative technologies of recent years, and has led to a revolution in personalized medication using medical devices [1
] and modified-release products [2
]. The first solid dosage form (Spritam) of a medication printed by 3D technology was approved by the FDA in 2015. Nowadays, 3D printing methods may support the development of personalized medicine and therapy, for example in cardiac and orthopedic surgeries [3
]. This innovative technique has also attracted significant attention in dental and plastic surgeries [4
]. There are other technologies that are also being used for biomedical applications; for instance, fused deposition modeling (FDM) or stereolithography (SLA) [5
]. Obviously, the first is used to prepare medical devices for surgical implantation, but is still onerous and highly expensive, whilst the second is cheap and suitable for prototyping devices both for biomedical analytical usage and clinical prototyping, such as drug patches [6
]. For both FDM and SLA, commercial polymers or pre-polymers are now widely available, but little or nothing is known about their behavior in terms of bacterial colonization or biofilm formation, and indeed whether the printing process itself has an effect on the bio-properties of the post-printed structures remains to be clarified.
The concept of 3D printing in the medical field is aimed at designing anatomically correct prototypes using different imaging techniques, such as MRI, CT, and so forth, in which these pictures are transformed into file formats for 3D printers [7
]. The possibility of producing individualized implants and oral drug formulations holds potential for a variety of different medical purposes. The use of 3D printing in the chemical sciences has expanded rapidly because different synthesis processes can be automatically controlled by, for instance, robot technics [8
]. Glatzel et al. reported that a robotic setup may produce 3D printed antibacterial assays, which may promote new opportunities in the development of laboratory practice [9
]. PLA-based implants are widely used due to their advantageous biodegradability and favorable cytocompatibility properties [10
]. They also have good thermal plasticity and suitable mechanical properties [11
]. Scaffold modifications/functionalizations have a great impact on the morphology and cytocompatibility of medical devices or 3D printed medicines. Side chain modifications may result in different surface properties [12
]. Similarly, the use of surface absorption of polymers may allow modulation of the chemical and biological properties of 3D printed scaffolds, as in the very recent paper of Thire et al. on the use of polydopamine absorption onto 3D printed PLA scaffolds [13
In a slightly earlier article, surface modification and the anti-microbial properties of the obtained systems were demonstrated. The previously utilized methods provided access to coated surfaces, however such systems can be expected to wear over time, which may limit their utility. Direct chemical modification of the polymers forming 3D printed objects would seem to be of interest, as the actual surface of the object is changed and not coated [14
Of the available methods to characterize printed and modified PLA objects, FT-IR will definitively show the presence of new chemical functions, but intensities of the IR bands may be weak in the case of surface changes. Contact angle measurements will provide information on the hydrophilicity and hydrophobicity of the surface, and when correlated with FT-IR allow rapid characterization of surface chemical modifications. Scanning electron microscopy is an appropriate method for characterization of porosity and pore size of the surface of implants. Values of positron lifetimes and the corresponding intensities measured by positron annihilation lifetime spectroscopy may be connected with the size and the structural defects of implants [15
To reach the general market with 3D printed implants, these devices must meet all standards and regulations defined by different authorities [16
]. Biocompatibility investigation is a compulsory test of implantable devices. The ISO 10993-5 standard determines the parameters of the required cytotoxicity test. The main requirement is the time of contact duration: In the case of limited exposure the duration time is less than 24 h, and for prolonged exposure 1–30 days of investigation is required. Contact periods of longer than 30 days is regulated as requiring long-term studies [17
]. Caco-2 cells are mainly used as a monolayer, rather than individual cells, however several assays are performed prior to reaching complete integrity, such as the end point or noninvasive cell viability assays (MTT assay, LDH test, real-time cell electronic sensing assay (RT-CES), etc.) [18
]. MTT assay is a broadly used, rapid colorimetric method to measure the in-vitro cytotoxicity of certain compounds on cell lines or primary cells. It is usually performed in 96-well plates, thus is a high throughput method, which excludes cell counting. For decades it was considered as evident that MTT is transformed in the mitochondria, however in the last few years, doubts have arisen regarding the mitochondrial localization of the formazan formation [19
Microbial biofilm formation could be a risk factor of implanted devices, potentially resulting in unpredictable complications [20
]. These microbial biofilm formations may contribute to causing an inflammatory response and result in subsequent operations, such as dental operations. The ability of Candida albicans
to form biofilms on medical devices could be a key property that enhances its ability to cause disease in humans [21
]. Here, the use of chemical modification may allow inhibition of microbial biofilm formation, or may enhance such surface colonization. Interestingly, both growth inhibition and enhancement can be of considerable use to the researcher. A key point in such a modification is the presence of reactive ester groups in many of the polymers used in 3D printing, thus polylactic acid (PLA) and polyethylene terephthalate (PET) are based on polymers with ester backbones, whereas poly(methyl methacrylate) (PMMA) has ester side chain functions.
Many studies have been published on 3D printed implants and dosage forms, however, the connections between the chemical structures, structural parameters, and cytocompatibility results have not been well characterized yet. The objectives of our study were to find connections between the abovementioned parameters in case of chemically modified PLA based 3D printed polyvalent test plates platforms, or PVTPs. Various amines were used for the post fabrication chemistry to form active amide functionalities along the surface of 3D printed PVTP structures.
The structural parameters of the samples were evaluated using Fourier transform infrared (FTIR) spectroscopy, contact angle measurements, scanning electron microscopy (SEM), surface roughness measurement, and positron annihilation lifetime spectrometry (PALS). A cytotoxicity test based on an MTT assay on Caco-2 cells and investigation of biofilm formation with Candida albicans were performed to certify the cytocompatibility of the PVTPs. The work is a first step towards chemical functionalization of FDM and SLA 3D printed objects and also the study of the cytocompatibility of generic structures, and shows that simple structures can be produced rapidly and cheaply by 3D printing for use in biological experiments.
Nowadays, 3D printing technologies for medical purposes are mainly focusing on bioprinting, dental applications, orthopedic applications, and development of modified release oral drug delivery systems [2
]. In our study, morphological characterization and in-vitro anti-fungal and cytocompatibility tests of chemically modified PLA-based 3D printed PVTPs are presented. In our research, surface modification of PLA polymer using oligoamines introduced better anti-infective properties to the polymer without altering its other properties, however in the paper by Tappa et al., addition of anti-infective agents was associated with a decrease in the mechanical properties of PLA, even though it retained its anti-infective properties [44
Polyvalent test plates were designed for investigation of the physical, chemical, and in-vitro biological properties of FDM printed materials, thus the term polyvalent in this article refers to the use of a single structure for testing of a wide range of properties. The advantage of such a PVTP was the exclusion of effects due to differing geometries, and enabling of the direct comparison between test results. A polyvalent can be used in many situations—chemistry, physics, mechanics, biology, and cell growth, and even biofilm formation.
The reason the printed PVTPs were chemically modified was to increase their favorable surface properties and biocompatibilities. These chemical modifications were verified by FT-IR spectroscopy, showing introduction of amide and amine functions. The FT-IR data was upgraded, highlighting the bands corresponding to the ester C=O, amide C=O, and N-H stretches. The intensities of the amide C=O and N–H are relatively weak as they are surface bonds. Other methods are able to show amide formation, but none are applicable to surface modification. For example, 13C NMR would not detect C at less than 0.1% [45
]. Other methods would not show the formation of covalent bonds.
There is clear evidence for the influence of surface topographical and wetting characteristics on macromolecular and cellular levels at implant interfaces [46
]. The polymer ester backbone was surface modified by a series of ramified and linear oligoamines to increase the hydrophilicity of the PVTP surfaces and introduce positive charge. These hydrophilic surface modifications were certified by contact angle measurement, which is crucial based on work by Song et al. because wettability of PVTPs may be affected by chemical modification [47
]. However, as ethylene diamine is a small and highly water soluble molecule, it would be expected to be washed off the surface. This is not the case, as the contact angle remains constant after washing vigorously. The surface wettability of biomaterials determines the biological events at the biomaterial/host interface [46
]. Wettability is modulated by surface chemistry, as was shown in our work and in the articles of Nasrin et al. and Mi et al., because a large range of solid characterization methods can be used for the qualification of different 3D printed polymers in these articles [28
]. There is also evidence regarding the pH dependence of the contact angle, but the method is as yet not fully proved [49
The roughness of our PLA based PVTPs was determined by scanning electron microscopy. However, the result of surface scanning depends on many parameters (i.e., the applied filters, the evaluation areas, etc.). The assessment of micron, submicron, and nano-roughness of different implant surfaces is inevitable. Nevertheless, there are only a few standardized methods for the correct determination of implant surfaces [46
], although the prediction of the biological performance of different implants in the human body is essential.
Positron annihilation spectroscopy (PALS) measures the supramolecular structure of polymeric-based implants, which can give correct information about the pore sizes and the porosity of the surfaces. Approximately the upper 100 μm of the surface can be examined with this method, and deviations can be seen in a nano-dimensional range. This method is based on one of Einstein’s principles, and the detected o-Ps lifetime values are in correlation with the free volume holes of the samples, which enables the comparison of the loaded and unloaded delivery bases. The latter could be useful in the characterization of amorphous solid dispersions or solutions [50
Comparing the results of PALS and SEM experiments, PLA-ED, PLA-TET, and PLA-Tris all showed high free volumes and the presence of numerous pits. For PLA-ED and PLA-Tris these were ca 1 µm in diameter, and for PLA-TET these pits were ca 100–200 nm in diameter. However, for MeNprN, few pits were present and the free volume was much reduced. Thus, there appeared to be a direct relationship between the free volume measured by PALS and the presence of large numbers of pits observed by SEM.
PLA showed good biocompatibility results and was biodegradable through hydrolytic degradation [47
]. Our samples were stored in DMEM medium at 37 °C until the end of the 12th day, and these extracts were measured on days four, eight, and 12 on the Caco-2 cells. The incubation period was 30 min. The cytotoxicity test was harmonized with the ISO 10993-5 standard, but the incubation period was shorter [41
]. There is clear evidence that PLA degrades into lactic acid and glycolic acid during storage, and different factors (pH, molecular weight, etc.) can influence this degradation [51
]. Many studies have dealt with the degradation of PLA polymers, so the main focus of our study was not the investigation of PLA degradation but the cytotoxic and biofilm formation effects of the modified PLA samples stored in DMEM medium, where the samples may degrade. In-vitro cytotoxicity methods can be the first filter in the assessment of cytocompatibility, because the assay is sensitive, efficiently adaptable, and well reproducible [52
]. MTT assay is a highly efficient screening test because only the viable cells are able to convert the dye through their mitochondrial enzymes. Van Tonder et al. revealed that the MTT dye alteration to formazan salt depends on the cells’ metabolic rate and number of mitochondria [19
]. In spite of this limitation of MTT assay, it is still the most reliable and quickest test for the assessment of cytocompatibility [53
]. Ramot et al. revealed that PLA can provoke an inflammatory reaction through implantation [54
]. Silva et al., however, certified our result because they pointed out that these inflammatory responses, as adverse reactions caused by PLA samples, are extremely rare [55
]. Based on the results of the MTT test our PLA samples proved to be cytocompatible.
Biofilm formation is also a compulsory element of cytocompatibility examination, based on the paper by Yang et al. In this research, 3D printed polylactide-co
-glycolide and hydroxyapatite PVTPs were examined [56
]. Biofilm formation can be associated with long-term implantation, which can lead to infections and antimicrobial resistance [34
]. During implantation any kind of infection could be dangerous, because it may result in serious inflammation and the rejection of implants. However, biofilm formation can alter the structure of PLA and may cause liberation of different APIs in the case of 3D printed medicine, for example [54
]. In our study, biofilm formation was performed with Candida albicans
SC5314 reference isolate, because one of the most common biofilm forming fungi are Candida
] and other species of Candida
can cause serious invasive candidiasis, with a mortality rate of about 45% [57
]. Ideally, the measured absorbance is zero, which means that no kind of biofilm is formed on the implant by the reference isolate. According to the evaluation of biofilm formation, the chemically modified PLA-PVTPs are biocompatible.
In our paper, we demonstrated the importance of surface characterization and cytocompatibility investigations of 3D printed PLA- PVTPs and chemically modified PLA-based PVTPs. The amines as side chains can favorably alter the surface properties, wettability, and biofilm formation of PLA PVTP. Low biofilm formation ability and favorable cytocompatibility profiles were also presented in the case of prolonged exposure. There are connections between the type and the other properties of PLA PVTPs. Overall, the properties of 3D printing materials can be dramatically enhanced by the modification of base polymers. These implants may ensure a high possibility for the incorporation of different antimicrobial APIs. According to these results, PLA-ED, PLA-Tris, and PLA-TET PVTPs can show favorable surface and anti-infective properties. Therefore, these samples were selected for further in-vivo and/or human studies.